2&#39;3&#39;-dideoxy-2&#39;,3&#39;-didehydro-7,8-disubstituted guanosines and their immunostimulative effect

ABSTRACT

Immunostimulating 7,8-disubstituted guanine derivatives that also contain a β-9,1&#39;-linked-2&#39;,3&#39;-dideoxy-2&#39;,3&#39;-didehydroribosyl substituent are disclosed whose structures are represented by Formula I ##STR1## wherein X is O or S; R 1  is a hydrocarbyl or substituted hydrocarbyl moiety having a length of about one to about seven carbon atoms; R 2  is hydrogen or C 1  -C 8  acyl; and the pharmaceutically acceptable base addition salts thereof. Also disclosed are compositions containing an immunostimulating guanine derivative and processes for using the same.

TECHNICAL FIELD

The present invention relates to immunostimulating compounds, and morespecifically to 7,8-disubstituted guanine derivatives that also containa β-9,1'-linked-2',3'-dideoxy-2',3'-didehydroribosyl substituent as wellas to compositions containing those derivatives and processes for theiruse.

BACKGROUND OF THE INVENTION

An animal's immune system is comprised of numerous elements thatcounteract, eliminate, or neutralize agents that are recognized by thatsystem as foreign to the animal host. Generally, but not necessarily,the agent or substance recognized as foreign by the immune system hasits origin exogenous to the animal host. Exemplary of such exogenoussubstances are infectious bacteria and the by-products of their cellularactivity, virus particles and their proteins, proteins injected byinsect stings, and the like. In autoimmune diseases, such as rheumatoidarthritis, the host animal's immune system perceives host-made proteins(self-made proteins) as if they were foreign.

The principal effectors of the immune system are the B and T leukocytes.The B lymphocytes mediate humoral immunity, whereas cytotoxic T cells,natural killer (NK) cells, and T cell mediators'of delayed-typehypersensitivity are principal effectors of cell-mediated immunity.

T cells express important surface antigens designated CD 2, 3, 4, 5 and8 that are related to T cell functions. Helper T cell precursors are ofthe CD 4⁺,8⁻, phenotype. Th₁ and Th₂ subsets of CD 4⁺,8⁻ T cellsnormally participate in the activation and regulation of B cells. Thesehelper T cells are known to assist in activation, differentiation andregulation of immunoglobulin-secreting B cells after antigen presentingcells, such as B cells, macrophages and dedritic cells, take up,process, and present antigen in association with class II MHC molecules.The Th₂ cells provide cell associated and soluble (LK) signals for Bcell proliferation and differentiation, including interleukins (IL)-4,5, 6 and 10. Th₁ cells secrete a different spectrum of regulatorycytokines, including IL-2 and interferon (IFN)-γ.

Guanosine-3',5'-cyclic monophosphate (cGMP) was previously thought to beimplicated as a naturally occurring agent for providing the requiredintracellular second message for B cell proliferation.8-Bromoguanosine-3',5'-cyclic guanosione monophosphate (8-Er-cGMP, ananalog of cGMP,) has been found to be a weak synthetic intracellular Blymphocyte mitogen.

The immune response can be modified by artificial suppression (hereincalled immunosuppression) or enhancement (immunopotentiation,immunostimulation or immuno-enhancement). Immunosuppression; i.e.,naturally or artificially induced decreased responsiveness, can beachieved by eight general methods: (1) administration of an appropriateform or amount of antigen, (2) administration of specific antisera orantibody, (3) use of other biologic reagents such as antilymphocyteantisera, (4) use of immuno-suppressive drugs or hormones, (5)radiation, and (6) surgical removal of lymphoid tissue, (7) aging (orsenescence) or certain heritable conditions, and (8) infection withcertain microbial agents. Immunopotentiation can be achieved byadministration of an agent effecting an increase in the rate at whichthe immune response develops, an increase in the intensity or level ofthe response, a prolongation of the response, or the development of aresponse to an otherwise non-immunogenic substance.

The agents that are known to enhance immune responses are generallytermed adjuvants. Based upon relative activities, adjuvants can beplaced into one of two general categories: (1) those providing generalpotentiation; i.e., substances that enhance both cellular and humoralimmune responses for a wide variety of antigens, and (2) those providingspecific potentiation; i.e., substances that enhance specific responsesto certain antigens only. Exemplary adjuvants include the followingcategories: (1) water and oil emulsions, e.g., Freund's adjuvant, (2)synthetic polynucleotides, (3) hormones, drugs and cyclic nucleotides,(4) endotoxins, (5) proteinaceous lymphokines and monokines such as theinterleukins and growth factors.

An example of a substance capable of specifically potentiating theimmune response is a transfer factor, a dialyzable leukocyte extract(DLE) obtained from human peripheral leukocytes. It has been reportedthat the transfer factor exhibits some effectiveness in patients withimmunodeficiencies and possible effectiveness in cancer patients and inpatients with limited immunodeficiencies. However, much remains to belearned about this particular substance.

In some diseases and physiological conditions such as X-linkedagammaglobulinemias, senescence and drug-induced-immunosuppression, Bcell activation and differentiation is lacking and/or exists only at areduced level, thereby lessening the immune response of the host. Thesediseases and conditions are representative of naturally and artificiallyinduced immunosuppressed states. Here, enhanced activation anddifferentiation, if it can be effected, tends to beneficially lessen thedisease manifestation and/or improve the patient's condition.

An immunopotentiated state can be illustrated by the bodily conditionafter vaccination. Here, the immune response, already enhanced due toantigenic stimulation could be beneficially enhanced still further toprovide an improved degree and/or duration of immunity by either asubsequent exposure to antigen, use of an adjuvant, or both.

Goodman and Weigle U.S. Pat. No. 4,539,205 describes modulation ofanimal cellular responses with 8-substituted guanine derivatives bonded9-1' to an aldose having 5 or 6 carbon atoms in the aldose chain (ring).The cellular modulations described in that patent relate mostly toimmunomodulation such as adjuvanticity in producing primary andsecondary immune responses. Activity against certain neoplasticconditions is also disclosed as are T cell-replacing activity, an IL-1like activity on thymocytes, and induction of the release of lysosomalenzymes from neutrophils. The 8-substituents in those molecules haveelectron withdrawing inductive effects relative to hydrogen. Thus, halo,mercapto or its thioxo tautomer, acyl mercapto, alkyl sulfido, nitro,cyano, keto, halomethyl and methyleneoxy alkyl and the like weredisclosed as useful, whereas electron donating substituents such as anamino group were found to be inactive.

U.S. Pat. No. 4,643,992 further discloses the use of derivatives of8-hydroxyguanine (8-oxoguanine), 7-methyl-8-oxoguanine and7-methyl-8-thioxoguanine in modulating animal cellular responses.Further results using guanine derivatives disclosed in U.S. Pat. No.4,539,205 are also disclosed in U.S. Pat. No. 4,643,992, as are similarresults using guanine derivatives disclosed for the first time in thatpatent.

Still further, several papers and book chapters have been published bysome of the present inventors and their co-workers relating to stillfurther effects of compounds disclosed and claimed in U.S. Pat. No.4,643,992. Exemplary of those published papers are Goodman, Proc. Soc.Exp. Biol. Med., 179:479 (1985); Goodman, J. Immunol., 136:3335 (1986);Goodman and Weigle in Purine Metabolism In Man, Part 3, Nyhan andThompson, eds., Plenum Press, New York, page 451 and 443 (1986); Goodmanand Weigle, J. Immunol., 135:3284 (1985); Goodman and Wolfert, Immunol.Res., 5:71 (1986); Goodman, J. Immunol., 137:3753 (1986); and Goodmanand Hennen, Cell. Immunol., 102:395 (1986).

U.S. Pat. No. 5,011,828 describes certain 7,8-disubstituted guaninenucleosides that enhance an immune response in human and animal cells.The nucleosides described in this patent are improvements over the otherguanosine derivatives mentioned previously in that they either provide asimilar response at a lower dose or provide a greater enhancement of theresponse at a given dose. The 7-substituents of these guanosinederivatives are straight, cyclic or branched chain hydrocarbyl radicalshaving a length greater than an ethyl group and less than a decyl group.

U.S. Pat. No. 5,093,318 describes further 7,8-disubstituted guaninenucleosides that are also immunostimulators. Those immunostimulators hadsimilar 8-substituents to the compounds of U.S. Pat. No. 5,011,828, buthad had heteroatom-containing substituents at the 7-position instead ofhydrocarbyl substituents.

The disclosure that follows describes 7,8-disubstituted guaninederivatives whose 9-substituent groups are not saccharide derivatives aswere the 1-ribosyl derivatives of either of U.S. Pat. Nos. 5,093,318 or5,011,828.

SUMMARY OF THE INVENTION

It has been found that N-9 2',3'-dideoxy-2',3'-didehydroribosylderivatives of 7,8-disubstituted guanines have immunoenhancing(immunostimulatory) properties. A contemplated compound corresponds instructure to the following general formula I: ##STR2## wherein X is O orS, most preferably O;

R¹ is a hydrocarbyl or substituted hydrocarbyl group (moiety or radical)having a length of one to about seven carbon atoms. More preferably, R¹is selected from the group consisting of (i) a C₁ -C₅ aliphatichydrocarbyl group, (ii) an aralkyl hydrocarbyl radical such as a benzylor phenethyl radical and (iii) a substituted aralkyl radical whosesubstituent is selected from the group consisting of halogen, nitro, C₁-C₂ alkyl, trifluoromethyl, amino and substituted amino, wherein theamine substituents are mono- or di-C₁ -C₂ alkyl, C₁ -C₃ acyl andsulfonyl groups;

R² is hydrogen or C₁ -C₈ acyl; and

the pharmaceutically acceptable base addition salts thereof.

Pharmaceutical compositions containing such compounds and methods ofenhancing immune response in human and animal cells using such compoundsare also disclosed.

DETAILED DESCRIPTION OF THE INVENTION A. Compounds

More particularly, a compound of the present invention is represented bythe general formula I: ##STR3## wherein X is O or S, most preferably O;

R¹ is a hydrocarbyl or substituted hydrocarbyl group (moiety or radical)having a length of one to about seven carbon atoms. More preferably, R¹is selected from the group consisting of (i) a C₁ -C₅ aliphatichydrocarbyl group, (ii) an aralkyl hydrocarbyl radical such as a benzylor phenethyl radical and (iii) a substituted aralkyl radical whosesubstituent is selected from the group consisting of halogen, nitro, C₁-C₂ alkyl, trifluoromethyl, amino and substituted amino, wherein theamine substituents are mono- or di-C₁ -C₂ alkyl, C₁ -C₃ acyl andsulfonyl groups; and

R² is hydrogen or a C₁ -C₈ acyl radical. A pharmaceutically acceptablebase addition salt of a compound of formula I is also contemplated.

The hydrocarbyl radical chain lengths are measured along the longestlinear carbon chain in the molecule. Such lengths can be readilydetermined by using published bond angles, bond lengths and atomicradii, as needed, to draw and measure a staggered chain, by buildingmodels using commercially available kits whose bond angles, lengths andatomic radii are in accord with accepted, published values, or by use ofwell known computer programs. Radical lengths can also be determinedsomewhat less exactly by assuming unsaturated bonds to have the samelength as saturated bonds and that bond angles for unsaturated bonds arethe same as those for saturated bonds, although the above mentionedmodes of measurement are preferred. Where a heteroatom is present, thatatom is considered to be a carbon atom for purposes of measuringsubstituent chain length. For example, a 4-nitrobenzyl substituent groupis considered to have a length of about seven carbon atoms.

Hydrocarbons and hydrocarbyl radicals contain only carbon and hydrogenatoms. Hydrocarbyl radicals useful as R¹ groups can broadly be dividedinto aliphatic and aromatic radicals. Aliphatic radicals include (i)saturated alkane (alkyl radicals) and (ii) mono- and polyunsaturatedalkenes and alkynes (alkenyl and alkynyl radicals), respectively.Cyclic, straight chain and branched chain radicals exist for each typeof aliphatic radical. Contemplated aromatic R¹ aralkyl radicals includearalkane, aralkene and aralkyne radicals that contain an aromatic ringlinked to an aliphatic group. Aromatic R¹ groups are benzyl or phenethylgroups and their derivatives. Exemplary R¹ hydrocarbyl radicals aredescribed below.

As used herein unless otherwise noted, an alkyl substituent groupincludes straight and branched chains such as methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl,2-methyl-3-butyl, 1-methylbutyl, 2-methylbutyl, neopentyl, hexyl andheptyl, as well as cyclic groups as are discussed hereinafter. Ofcourse, if the alkyl substituent is branched there must be at least 3carbons. The group of R¹ C₁ -C₅ alkyl radicals is more preferred.

Unsaturated radicals constitute yet another group of aliphatic radicals.Exemplary radicals include allyl, 3-butenyl, 2-methyl-3-butenyl andbutadienyl. C₃ -C₅ Beta-alkenyl radicals are a particularly preferredgroup of unsaturated hydrocarbyl radicals. C₃ -C₅ Beta-alkenyl radicalsare a particularly preferred group of unsaturated hydrocarbyl radicals.C₁ -C₅ Beta-alkenyl radicals contain an ethylenic double bond beta tothe 7-nitrogen atom of the guanosine. Exemplary radicals include allyl(2-propenyl), 2-butenyl, 2-pentyl and 3-methyl-2-butenyl. Allyl is themost preferred R¹ group.

Cyclic aliphatic groups include those compounds having 5-7 carbon atomsin the cyclic ring. Exemplary groups include cyclopentyl, cyclohexyl,cycloheptyl, 3-methylcyclopentyl, 4-methylcyclohexyl, and the like.Monoethylenically unsaturated cyclic aliphatic groups are alsocontemplated such as cyclopent-2-enyl and cyclohex-2-enyl groups.

Aralkyl radicals constitute another group of hydrocarbyl, R¹, radicals.Those radicals contain an aromatic ring, and include benzyl andphenethyl. Benzyl is a particularly preferred R¹ radical.

An above-discussed aralkyl hydrocarbyl group or radical can also besubstituted by one or more atoms other than carbon and hydrogen. Such asubstituted radical is referred to herein as a heteroatom-substitutedaralkyl hydrocarbyl group or radical.

Substituted aralkyl radicals are also contemplated R¹ radicals.Contemplated aralkyl radicals (benzyl and phenethyl radicals) can besubstituted on their phenyl rings with one or two, preferably one, C₁-C₂ alkyl or heteroatom-substituent selected from the group consistingof nitro, halogen, trifluoromethyl, amino and substituted amino whereinthe amine substituents are mono- or di-C₁ -C₂ alkyl and mono-C₁ -C₂acyl.

Substituents on a benzyl or phenethyl group that are electronwithdrawing groups relative to hydrogen by resonance or inductive effectas are discussed in Hine, Physical Organic Chemistry, 2nd ed.,McGraw-Hill Book Co., New York, pages 85-93 (1962) such as cyano, nitroand halogen are preferred. A 4-nitrobenzyl radical is a particularlypreferred radical.

Exemplary heteroatom-substituted aralkyl groups include 3- and 4-halo(fluoro, chloro, bromo and iodo) benzyl and phenethyl groups,3,4-dihalobenzyl such as a 3,4-dichlorobenzyl group, 3- and4-nitrobenzyl and phenethyl groups, 3- or 4-trifluoromethylbenzylgroups, 2-, 3- or 4-aminobenzyl or phenethyl groups, and their N-formylor N-acetyl (C₁ -C₂ acyl), or mono- and dimethyl or mono- and diethylderivatives. Exemplary C₁ -C₂ alkyl-substituted aralkyl groups include2-, 3- or 4 -methylbenzyl or phenethyl groups, a 3,4-dimethylbenzylgroup. Aralkyl groups substituted at the 3- or 4-position of the phenylring are preferred. Di-C₁ -C₂ alkyl- or C₁ -C₂ acyl-amino-substitutedaralkyl groups are preferred over those having one or no substituent onthe amine nitrogen atom. A heteroatom-substituted benzyl group is apreferred substituted aralkyl group.

When R² is hydrogen, the 2',3'-dideoxy-2',3'-didehydroribosyl(dideoxy-didehydro) groups is unsubstituted. That unsubstituted ring canalso be named as a 1-(2,3-dideoxy-β-D-glyceropent-2-enofuranosyl) group,radical or moiety. The dideoxydidehydro ring substituent hydroxyl groupcan also be acylated (esterified) with a C₁ -C₈ acyl moiety, group orradical. Exemplary C₁ -C₈ acyl groups include formyl, acetyl, propionyl,iso-butyryl, hexanoyl, octanoyl, cyclohexylcarbonyl, benzoyl, andtoluoyl (toluyl).

A contemplated C₁ -C₈ acyl group can be added to the formeddideoxy-didehydro ring after the formation of a compound of formula I inwhich R² is hydrogen by a well known esterification reaction. Thus, a C₁-C₈ acyl anhydride such as acetic anhydride or a C₁ -C₈ acyl halide suchas benzoyl chloride can be used as the acylating agent in an aproticstandard solvent such as pyridine.

A contemplated guanosine is substantially free from ionic charge atphysiological pH values; i.e., about pH 7.0 to about pH 7.5, except forthe ionic charges that might be provided by the relatively acidic1-position ring nitrogen atom. Thus, a useful molecule is free of acidand base-containing moieties that are not present in guanosine such asphosphate, carboxylate or sulfonate moieties and alkylamines. Freedomfrom ionic charge can be assayed by a lack of relative movement at pH7.0-7.5 in an electrophoretic study. An amine substituent whose pK_(a)value is about 6.0 or less can be present as where an R¹ group is ananiline derivative. Substantially all carboxylate, phosphate, sulfate orsulfonate substituents bear an ionic charge at physiological pH values.

Many contemplated guanines are themselves weak acids, and as such canform base addition salts. Such salts are useful in providing storagestability and do not provide an added ionic charge to a guaninederivative used in a method of the invention because of the bufferingeffect provided by the host's blood and lymph systems or the buffer of aculture medium.

Pharmaceutically acceptable, non-toxic base addition salts of guaninederivatives are useful herein, and can be formed by treatment of theimmune response-enhancing agent with an appropriate base, in a suitablesolvent such as water or a lower alkyl alcohol such as methanol orethanol. Exemplary inorganic bases include sodium hydroxide, potassiumhydroxide, ammonium hydroxide and the like. Exemplary organic basesinclude tris-(hydroxymethyl)-aminomethane (TRIS),4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) and thelike. Conversely, the base addition salt form can be converted to thefree guanosine form by treatment with acid.

A contemplated compound can be synthesized by many well known methods.Most preferably, a contemplated compound is prepared from acorresponding guanosine derivative such as Compound 1, whose synthesesare disclosed in U.S. Pat. Nos. 5,011,820 and 5,093,318, following thereaction set out in Scheme 1, below, that illustrates synthesis ofCompound 3. ##STR4##

As shown, the reaction comprises reacting a 7,8-disubstituted guanosineCompound 1 with 1-acetoxyisobutyryl bromide (c.f. Russel et al., J. Am.Chem. Soc., 95:4025-4030 (1973) to give a mixture of vicihalbromoacetates, which were than directly reduced with zinc powder to giveCompound 2, in which there is unsaturation between the 2' and 3' carbonsof the sugar ring. The first reaction with 1-acetoxyisobutyryl bromidewas carried out in acetonitrile at reflux for about 30 minutes. Thereaction requiring the zinc powder is carried out in alcoholic solvent(e.g. EtOH) in the presence of small amounts of acetic acid, andrequires 1-3 days to achieve completion. Compound 2 is then treated withsodium methoxide in methanol to remove the 5'-acyl protective group, toobtain alcohol Compound 3. The resulting Compound 3 can then be reactedwith a suitable electrophile such as an acid chloride to effectacylation on the 5'-hydroxyl (R² =C₁ -C₈ acyl).

Exemplary compounds of this group preparable from the compounds of theabove U.S. patents are shown in Table 1, below.

                  TABLE 1                                                         ______________________________________                                         ##STR5##                      I                                              R.sup.1           Patent   Example                                            ______________________________________                                        Ethyl             5,011,828                                                                               6                                                 Propyl            5,011,828                                                                               5                                                 Allyl             5,011,828                                                                              8,9                                                Butyl             5,011,828                                                                               2                                                 2-Butenyl         5,011,828                                                                               3                                                 Benzyl            5,011,828                                                                              23                                                 Hexyl             5,011,828                                                                               4                                                 Carbethoxymethyl  5,093,318                                                                               2                                                 2,3-Dihydroxypropyl                                                                             5,093,318                                                                              13                                                 4-Nitrobenzyl     5,093,318                                                                              16                                                 2-Chloroethyl     5,093,318                                                                              18                                                 ______________________________________                                    

B. Compositions

A contemplated composition comprises a diluent amount of aphysiologically tolerable carrier (also referred to herein as a vehicleor diluent) admixed with an immunopotentiating (immuneresponse-enhancing or immunostimulating) effective amount of asubstituted guanine nucleoside derivative or salt described before.

A composition for in vivo administration is typically provided for peroral or parenteral administration in customary unit dosage compositions.The term "unit dosage" and its grammatical equivalents as used hereinrefer to physically discrete units suitable as unitary dosages for humanpatients and other mammals, each unit containing a predeterminedeffective amount of the guanosine analog derivative active ingredientcalculated to produce the desired therapeutic effect in association withthe required physiologically tolerable carrier, e.g. a diluent or avehicle. The specifications for the unit dosage forms of this inventionare dictated by and are directly dependent on (a) the uniquecharacteristics of the active guanosine analog derivative ingredient andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active ingredientfor therapeutic use in vitro, as well as in vivo in humans and otheranimals.

Examples of suitable unit dosage forms in accord with this invention aretablets, capsules, pills, powder packets, granules, wafers, and thelike, segregated multiples of any of the foregoing, as well as liquidsolutions, emulsions and suspensions. Liquid compositions can beadministered in usual manners such as subcutaneously, intraperitoneally,intramuscularly, intravenously perorally or the like.

The amount of active ingredient that is administered in vivo as aneffective immunostimulating amount depends on the age and weight of thepatient, the particular condition to be treated, the frequency ofadministration, and the route of administration. The total daily doserange can be about 0.01 to about 400 milligrams per kilogram of bodyweight, more preferably about 0.1 to about 400 milligrams per kilogramof body weight. The human adult dose is in the range of about 70 toabout 7000 milligrams daily, given either as a single dose or in 3 or 4divided doses. Veterinary dosages correspond to human dosages with theamounts administered being in proportion to the weight and metabolicrate of the animal as compared to adult humans.

It will be appreciated by those skilled in the art that useful in vivoconcentrations can vary from animal species to animal species. Thoseskilled workers also know that appropriate concentrations can be readilydetermined.

Concentrations for the in vitro contacting of animal cells are about1×10⁻⁶ molar to about 10⁻³ molar for cell concentrations of about 10⁶⁻¹⁰⁷ cells per milliliter. More preferably, the concentration is about1×10⁻⁵ molar to about 3×10⁻⁴ molar. The peak concentration; e.g., theconcentration that provides the greatest adjuvanticity, mitogenicity orNK cell activation, for a given guanosine analog can vary as much as tenor more fold when studied in mouse as compared to human lymphocytesystems.

A composition can be solid or liquid. Physiologically tolerable carriersare well known in the art. Exemplary of liquid carriers are sterileaqueous solutions that contain no materials in addition to the activeingredient guanine derivative and water, or contain a buffer such assodium phosphate at physiological pH value, physiological saline orboth, such as phosphate-buffered saline. Still further, aqueous carrierscan contain more than one buffer salt, as well as salts such as sodiumand potassium chlorides, dextrose and other solutes. The latter carriersare exemplified by Ringer's Injection, Dextrose Injection, Dextrose andSodium Chloride Injection and Lactated Ringer's Injection. Preferably,the solution should be isotonic unless it is an oral pediatricsuspension.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional phases areglycerin, vegetable oils, such as sesame oil, cotton seed oil, andwater-oil emulsions.

Exemplary solid carriers include those materials usually used in themanufacture of pills, tablets or capsules, and include corn starch,lactose, dicalcium phosphate, thickeners such as tragacanth gum andmethylcellulose U.S.P., finely divided SiO₂, polyvinylpyrrolidone,magnesium stearate and the like. Additionally, the solid carrier caninclude biodegradable and non-biodegradable polymers, polypeptidecarriers, affinity carriers such as AFFI-GEL 601 (phenyl boronate resinavailable from BIO-RAD Laboratories, Richmond, Calif.), liposomes andsynthetic polymers, as are known in the art. Antioxidants such asmethylparaben and propylparaben can be present in both solid and liquidcompositions, as can sweeteners such as cane or beet sugar, sodiumsaccharin, sodium cyclamate and the dipeptide asparticphenylalaninemethyl ester sweetener sold under the trade name NUTRASWEET® (aspartame)by G. D. Searle Co.

C. Processes of Treatment

A process for enhancing the immune responses of leukocytes is alsocontemplated. Preferably, the immune response is an antigen-specificresponse. In accordance with this method, leukocytes such as lymphocytepreparations, B cells, T cells, NK cells, LAK cells, neurophils andmacrophages are contacted separately or in combination in an aqueousmedium with a before-described composition containing animmunostimulating effective amount of a before-described guaninenucleoside derivative.

The process can be practiced in vivo in humans, laboratory mammals suchas mice, rats and guinea pigs or in veterinary animals and pets such aspigs, horses, cattle, dogs and cats. The process can also be practicedin vitro in cell cultures such as in hybridoma culture for theproduction of monoclonal antibodies.

The leukocytes are contacted in an aqueous medium regardless of whetherthe composition of guanosine derivative is itself a solid or liquid, orwhether or not the liquid of the composition is aqueous. For the in vivoprocess, the aqueous medium is supplied at least in part by the water ofthe blood or lymph. For in vitro process, the aqueous medium is suppliedat least in part by the culture medium used.

Contact between the composition and leukocytes is maintained for a timeperiod sufficient for the contacted cells to manifest the enhancement oftheir immune response. The immunostimulation can itself be manifest incellular proliferation, enhanced antibody secretion, enhanced T helperactivity, enhanced cytotoxic activity, enhanced cytokine production fromT cells, B cells, NK cells and macrophages, enzyme secretion fromneutrophils, and the like, as are well known.

The specific results discussed hereinafter illustrate a non-specificmitogenic response of murine spleen B cells, as well as the preferred NKcell-mediated cytotoxicity and antigen-specific antibody responses ofmurine B cells that can, but need not be, depleted of T cells.Additional illustrative antigen-specific immunoenhancements that can beachieved using a process of the invention include proliferation of Tcells, the in vitro reconstitution of the primary and anamnestic immuneresponses in murine or human immunodeficient B cells, T cell-replacingactivity in normal or immunodeficient murine or human B cells, and an invivo enhancement of murine antibody production.

For use in vivo, contact between leukocytes and a composition istypically maintained for a time period sufficient for the animal toclear the guanine derivative from its body as by metabolism, excretionor both processes. That time period can be longer than that required forimmunostimulation to be manifest. Contact with an individual unit doseis typically maintained for a time period of hours to about a week ormore, depending, for a given compound, upon the carrier or vehicle used.

Contact in vitro can be maintained for a period of time sufficient forone of the before-described immunostimulations to become manifest asdetermined by standard assay techniques. Such maintenance timestypically take about one to about seven days of time, and more usuallyabout 2 to about 6 days.

A contemplated compound was assayed to determine its ability to enhancethe immune response. The assays carried out are described hereinafter.In each of the assays, 7-allyl-8-oxoguanosine (loxoribine) was used as astandard. The results of each of the assays are reported in Table 2 thatfollows the description of the assays.

As will be seen from the results shown in Table 2, a contemplatedcompound exhibited substantially the same potency in activating NKcells, slightly less mitogenic potency and about one-half theadjuvanticity as were exhibited by loxoribine. Thus, using Compound 3,for example, one can induce NK cell activation about equal to thatobtained by loxoribine while limiting a side reaction caused byloxoribine.

NK ASSAY A. Animals

Male CBA/J and C3H/HeJ mice were obtained from Jackson Laboratories, BarHarbor, Me. All mice were used at 8 to 12 weeks of age. They were fedPurina rodent laboratory chow and tap water ad libitum.

B. Tissue Culture Medium

Culture medium was RPMI 1640 medium (flow Laboratories, McLean, Va.)supplemented with 5 percent fetal bovine serum (FBS, GIBCO, GrandIsland, N.Y. or HyClone Laboratories, Logan, Utah) and 100 IU/mlpenicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (all from FlowLaboratories, McLean, Va.).

C. Cell Lines

The NK-sensitive cell line YAC-1, was originally obtained from Dr. JohnRoder, Mount Sinai Hospital, Toronto, Ontario. It was maintained inbasic culture medium in a 37° C., 5 percent CO₂ incubator. Cell lineswere tested periodically for mycoplasma and found to be negative.

D. Loxoribine (7-allyl-8-oxoguanosine)

For these in vitro experiments 10.2 mg, loxoribine (provided by the R.W. Johnson Pharmaceutical Research Institute) were dissolved in 0.1 ml1N NaOH. This was further diluted with 9.9 ml culture medium to providea 3 mM stock solution. The stock solution was filtered through 0.22 mMmillipore filters and diluted further with sterile medium. Vehiclecontrols contained equivalent concentrations of NaOH.

E. Lymphoid Cell Preparation

Mice were killed by cervical dislocation and spleens were removedaseptically and pressed through a stainless steel mesh. Cells werecentrifuged for 10 minutes at 220×g and resuspended in culture medium at10⁷ cells/ml.

F. In Vitro Culture of Lymphoid Cells with Loxoribine

Spleen cells at 4 concentrations, each in triplicate (10⁶, 5×10⁵,2.5×10⁵, 1.25×10⁵ spleen cells) were cultured in 96 well, round bottomculture trays (Linbro, Flow Laboratories, McLean, Va.) in a volume of100 μl. Compounds were added to the wells in a volume of 100 μl to giveappropriate final concentrations such as 3, 10, 30, 100, 300μM of drug.Cultures were incubated for 18 hours at 37° C. in 5 percent CO₂.

G. ⁵¹ Chromium Release Assay

YAC-1 target cells (5×10⁵) were incubated with 100 μCi sodium ⁵¹ Cr(Amersham Canada, Oakville, Ontario) for 1 hour at 37° C., in 5 percentCO₂, washed three times with Phosphate Buffered Saline (PBS), andsuspended in culture medium at 5×10⁴ /ml. The plates containing cells tobe assayed for cytotoxic activity were centrifuged at 220×g for 10minutes, 100 μl supernatant were removed from each well, and 100 μltarget cells were added to the wells. The plates were centrifuged for 5minutes at 150×g, incubated at 37° C. in 5 percent CO₂ for 4 hours, andcentrifuged for 5 minutes at 300×g. Supernatants were removed from thewells using the Skatron Supernatant Collection System (Skatron,Sterling, Va.) and the samples were counted in a Beckman gamma counter.Maximum lysis was obtained by counting the total CPM in 5×10³ targetcells. Minimum release, obtained from target cells cultured with mediumalone, was always lower than 10 percent of the maximum CPM. PercentLysis was calculated as: ##EQU1##

Each time a compound was assayed, loxoribine was assayed as well fordirect comparison purposes. The ED₅₀ value, taken as a measure ofpotency, varied from 15-34μM for loxoribine (see Table 2). The maximalresponse for loxoribine was assigned the value of 100 percent. Theactivity for other compounds is given in Table 2 as a direct comparisonto that of loxoribine.

MURINE ADJUVANTICITY In Vitro Adjuvanticity

Contacting animal antibody-producing cells with a composition usefulherein provides an adjuvant effect on the primary antibody response toSRBC (sheep red blood cells) and other immunogens when evaluated invitro. The immune response-stimulating composition and effective amountof immunogen, SRBC, are typically admixed to contact the cellssubstantially simultaneously. The words "antigen" and "immunogen" areused interchangeably here.

At optimal concentration, a composition containing an effective amountof a useful guanosine analog derivative enhances the response to antigenby at least about 4-25-fold. The effect is dose dependent. Enhancementof the antibody response cannot be accounted for by the additive effectsof the specific response to antigen and the polyclonal response to theguanosine analog derivative.

The adjuvant effect of compositions containing a useful guanosine analogderivative is exerted on immunogen-experienced (primed) as well as onnaive cells. Both responses are enhanced by contacting the cells withcompositions containing an effective amount of guanosine analogderivative. This adjuvant effect is dependent upon the concentration ofimmunogen added to culture. Thus, the primary IgM as well as thesecondary IgM and IgG responses to immunogen (antigen) are augmented bycontacting B cells with a composition containing an effective amount ofa guanosine analog derivative as active ingredient, and maintaining thatcontact as discussed herein.

Whereas immune responses; i.e., responses of B lymphocytes (or B cells)are observed to be enhanced at all immunologically effective doses ofimmunogen administered in an appropriately supportive cellular milieu,the degree of enhancement is usually greatest at optimal or near optimalimmunogen concentrations. Additionally, adjuvanticity of guanosineanalog derivatives is synergistic with immunogen and not simply due tothe sum of independent immunogen-specific and polyclonal (nonspecific)responses, as described above for primary antibody responses.

In summary, an enhanced immune response can thus be obtained bycontacting B cells substantially simultaneously with an effective,priming amount of immunogen and an immune response-enhancing compositionuseful herein, followed, after a primary immune response is obtained, byan additional contacting of the primed cells with a further effectiveamount of immunogen (antigen) alone or substantially simultaneously witha further amount of immune response-enhancing composition.

Guanosine analog derivative-containing compositions useful herein arethought to enhance the primary humoral immune response by actingdirectly upon the B cell and/or the immunogen-presenting cell. Thus, oneof these derivatives enhances the antibody response mounted againstT-independent antigens; i.e., responses that involve B cells andimmunogen-presenting cells. In addition, compositions containing aguanosine analog derivative can replace the need of B cells for T helpercells, as discussed hereinafter, and therefore exert their adjuvanteffect in cultures initiated in the substantial absence of intact,functional T cells. A replacement of T cells with T cell helper activitycontained in mixed lymphocyte culture (MLC) supernates or otherlymphokines that can be derived from T cells does not diminish theability of a guanosine analog derivative to augment the antibodyresponse.

Still further, the synergy observed between the soluble T cell signalcontained in MLC supernate and the guanosine analogderivative-containing composition acting on antigen-stimulated B cellsindicates that the signal supplied by each is qualitatively distinct.This synergy is observed over a range of supernate concentrations,indicating that the guanosine analog derivative is not simply providingmore of the same "signal" that T cells provide. A comparable degree ofsynergy can be observed when such B cell cultures are supplemented withT cells rather than with T cell-like supernates (which are in fact Tcell derived), and are contacted in the presence of immunogen with aguanosine analog derivative-containing composition useful in thisinvention.

In summary, T cell-mediated effects of the adjuvanticity of guanosineanalog derivatives are not ruled out by the observation ofT-independence for that adjuvanticity, i.e., the existence of a Tcell-independent phase. Thus, more substantial enhancement can beobserved from a composition containing the guanosine analog derivativeunder conditions of stimulation with T-dependent and T-independent type2 antigens (T cell dependent situations) than with T-independent type 1antigens (more completely T cell-independent), which suggests thepresence of a T cell-dependent component. Moreover, guanosine analogderivatives are thought to act (either directly or indirectly) onprecursors of T helper cells to increase the ability of a population ofsuch cells to support (i.e., help) an antibody response to immunogen.

Lymphocyte cultures

The serum-containing culture medium was prepared to contain thefollowing per 100 milliliters: 91.9 milliliters RPMI 1640 (FlowLaboratories, Inc., Rockville, Md.), 0.1 milliliters of 100×glutamine,1.0 milliliter of 100×sodium pyruvate, 1.0 milliliter of 50×nonessentialamino acids, 1.0 milliliter of water containing 10⁴ units of penicillinG and 10⁴ micrograms of streptomycin, and 5.0 milliliters of asupportive lot of fetal calf serum (FCS). These ingredients were admixedto apparent homogeneity. Spleen cell suspensions and populationsenriched for splenic B cells were prepared as described in Goodman etal., J. Immunol., 121:1905 (1978).

For evaluation of the primary humoral immune response to sheeperythrocytes (SRBC), 5×10⁶ to 10⁷ murine spleen cells were cultured in1.0 milliliter of 5 percent FCS-containing medium for 4 or 5 days in thepresence and absence of immunogen. Cells were incubated in culture trays(Costar, Cambridge, Mass.) at 37° C., in a humidified atmosphere of 10percent CO₂ in air using tissue culture boxes (CBS Scientific, Del Mar,Calif.) that were rocked at a frequency of 7 cycles per minute. PooledSRBC are available from the Colorado Serum Co., Denver, Colo.

Mice

CBA/CaJ mice, 8-16 weeks of age, are purchased from the JacksonLaboratory, Bar Harbor, Me. A breeding nucleus of CBA/N mice wasprovided by the Animal Production Section, National Institutes ofHealth, Bethesda, Md. SJL, BDF₁ and C₅₇ BL/6J mice 8-16 weeks old wereobtained from the mouse breeding facility at Scripps Clinic and ResearchFoundation, La Jolla, Calif. All mice were maintained on Wayne Blox F6pellets (Allied Mills, Inc., Chicago, Ill.) and chlorinated wateracidified with HC₁ to a pH value of 3.0

Cell Preparations

Spleen and thymus cell suspensions were prepared as described in Goodmanet al., J. Immunol., 121:1905 (1978). B cell-enriched populations wereprepared by treating 10⁸ spleen cells with a 1:1000 dilution ofmonoclonal anti-Thy 1.2 antibody (New England Nuclear, Boston, Md.) for30 minutes at 4° C. Treated cells were centrifuged at 280×gravity for 10minutes, antibodies were removed, and the cells were resuspended in a1:6 dilution of CBA RBC-absorbed guinea pig complement at 37° C. for 45minutes. Cells were then washed and cultured as described before.

The mouse adjuvanticity assays using a contemplated substituted guaninederiviative and similar control assays using loxoribine were carried outon the same days.

MITOGENESIS ASSAY Lymphocyte cultures

Murine spleen cells were cultured in microculture plates (No. 3546,Costar, Cambridge, Mass.) at a cell density of 4×10⁶ viable cellsmilliliter in a volume of 0.1 milliliter, together with incrementalconcentrations of assayed compounds. Microcultures were incubated at 37°C. in a humidified atmosphere of 5 percent CO₂ in air. Cultures were feddaily with 8 microliter of nutritional cocktail, Mishell and Dutton, J.Exp. Med., 126:423 (1967).

Measurement of DNA synthesis. During the final 24 hours of culture,cells were radiolabeled with 1.0 micro Ci of [³ H]TdR/culture (5 Ci/mM,Amersham Radiochemicals, Amersham, England). The microcultures wereharvested with a PhD cell harvester, Model 200A (Cambridge Technology,Inc., Cambridge, Mass.) onto glass fiber filter strips. Filter diskswere transferred to plastic scintillation vials, covered with liquidscintillation cocktail, and counted in a Beckman LS-230 liquidscintillation counter.

Because there is some variability between runs of the same compound,such as loxoribine, some of the data in Table 2 are presented as rangesof numbers where multiple experiments were conducted. The maximalresponse of loxoribine is represented by 100 percent, and the maximalresponse of the assayed compound is given relative to that value. Inaddition, the ED₅₀ values are given for loxoribine and the analog listedas part of this invention.

                  TABLE 2                                                         ______________________________________                                         ##STR6##                                                                              NK Activation          Murine                                        Compound (Potency)   Mitogenicity                                                                             Adjuvanticity                                 ______________________________________                                        3        89-99%       84%       454 (6 μM)                                          (11-12 μM)                                                                             (30 μM)                                               Loxoribine                                                                             100%        100%       100% (3-20 μM)                             (standard)                                                                             (15-34 μM)                                                                             (10-50 μM)                                            ______________________________________                                    

Example 1:2-Amino-7-(2-propenyl)-9-[1-(2,3-dideoxy-β-D-glyceropent-2-enofuranoxyl)]-purine-6,8(1H)-dione(Compound 3)

A suspension of 7-allyl-8-oxoguanosine (5 g, 14.8 mmol) in 20 mL of CH₃CN was treated with 1-acetoxyisobutryl bromide (15.5 g, 74 mmol). Themixture was heated at reflux for 30 minutes. The heat was removed, thesolution was cooled, and then it was treated with saturated aqueousNaHCO₃ and CH₂ Cl₂. The CH₂ Cl₂ layer was dried (MgSO₄), filtered andconcentrated. The residue was purified on a large dry silica gel column(CH₂ Cl₂ / MeOH, 96:4) to give a rust-colored solid of the expectedmixture of vicinal 2',3'-bromoacetates (7.5 g, 90 percent), slightlyunstable at room temperature. The majority of this material wasdissolved in EtOH (80 mL) and treated with Zn dust (9 g) and HOAc (0.5mL). As the conversion was slow at room temperature, after four hours,an additional 3 g of the Zn dust was added and the solution was heatedat 40° C. After 40 hours, the suspension was filtered through a coursefilter, then a Millipore filter and concentrated.

This residue was then treated with toluene and evaporated, dissolved inMeOH (10 mL), and treated with 25 percent NaOMe/MeOH (10 mL). Afterstirring overnight (about 18 hours), TLC showed the major formation of alower spot. Acetic acid (about 4 mL) was added, and the product wasextracted into CHC₁ ³ from water. The CHCl₃ layer was dried (MgSO₄),filtered and concentrated. It was then purified on 200 g silica (CH₂ Cl₂/MeOH/ether, 94/6) from which 1.7 g of nearly pure product wereisolated. Two recrysallizations from first CH₂ Cl₂ /ether and thenMeOH/benzene gave 750 mg of Compound 3. The compound was dried undervacuum overnight at 60° C. which caused about 2 percent of a lowerimpurity spot to form by TLC, presumably due to thermal cleavage of theglycosidic bond; m.p. 122° C., softening; 136°-144° C. (dec.). [α]_(D)²⁵ 74.1 (C 0.46, H₂ O). IR (KBr) 3342, 1680, 1633, 1597, 144 cm-1. Cl-MS(NH₃) :m/e 306 (M+1, 5 percent), 208 (100 percent) . ¹ H NMR (D₂ O) δ3.7 (br s, 2H) , 4.33 (s, 2H), 4.8-4.9 (m, 2H), 5.02 (d, 1H), 5.8 (m,1H), 5.91 (d, 1H), 6.17 (d, 1H), 6.62 (s, 1H), 7.24 (s, ca. 1H). The 1HNMR indicated the presence of a small amount of benzene ofrecyrstallization which was not removed during the drying. Anal (C₁₃ H₁₅N₅ O₄.0.3PhH.0.3H₂ O): C, 53.29; H, 5.25; N, 20.96. Found: C, 53.46; H,5.43; N, 20.74.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

What is claimed:
 1. A compound of the formula I: ##STR7## wherein X is Oor S; R¹ is a hydrocarbyl or a substituted hydrocarbyl moiety having alength of about one to about seven carbon atoms;R² is hydrogen or C₁ -C₈acyl; and the pharmaceutically acceptable base addition salts thereof.2. The compound of claim 1 wherein R¹ is selected from the groupconsisting of (i) a C₁ -C₅ aliphatic hydrocarbyl group, (ii) an aralkylhydrocarbyl radical and (iii) a substituted aralkyl radical whosesubstituent is selected from the group consisting of halogen, nitro, C₁-C₂ alkyl, trifluoromethyl, amino and substituted amino, wherein theamine substituents are mono- or di-C₁ -C₂ alkyl, C₁ -C₃ acyl andsulfonyl groups.
 3. The compound of claim 1 wherein R² is hydrogen. 4.The compound of claim 1, wherein X is O.
 5. The compound of claim 2wherein R¹ is a C₁ -C₅ aliphatic hydrocarbyl group.
 6. A compound of theFormula I ##STR8## wherein X is O or S; R¹ is a hydrocarbyl orsubstituted hydrocarbyl group having a length of one to about sevencarbon atoms that is selected from the group consisting of (i) a C₁ -C₅aliphatic hydrocarbyl group, (ii) an aralkyl hydrocarbyl radical and(iii) a substituted aralkyl radical whose substituent is selected fromthe group consisting of halogen, nitro, C₁ -C₂ alkyl, trifluoromethyl,amino and substituted amino, wherein the amine substituents are mono- ordi-C₁ -C₂ alkyl, C₁ -C₃ acyl and sulfonyl groups;R² is hydrogen or C₁-C₈ acyl; and the pharmaceutically acceptable base addition saltsthereof.
 7. The compound of claim 6 represented by the formula ##STR9##8. A pharmaceutical composition comprising diluent amount of aphysiologically acceptable carrier admixed with an immunostimulatingeffective amount of a compound of claim
 1. 9. A method of enhancing animmune response that comprises contacting leukocytes in an aqueousmedium with an amount of a compound of claim 1 sufficient to enhance theimmune response of those leukocytes.
 10. The method of claim 9 whereinthe cells are contacted in vitro in a culture medium.
 11. The method ofclaim 9 wherein the cells contacted include B cells.
 12. The method ofclaim 11 wherein the B cells are human B cells.
 13. The method of claim9 wherein the cells contacted include NK cells.
 14. The method of claim13 wherein the NK cells are human NK cells.
 15. The method of claim 9wherein the cells are T cells.
 16. The method of claim 9 wherein thecells are neutrophils.
 17. The method of claim 9 wherein the contactingis carried out in vivo by administering one or more unit doses of thecomposition to a mammal.